SYNERGISTIC ACTIVATION OF PTDINS 3-KINASE BY TYROSINE-PHOSPHORYLATED PEPTIDE AND BETA-GAMMA-SUBUNITS OF GTP-BINDING PROTEINS

Stimulation of differentiated THP-1 cells by insulin led to rapid accu mulation of PtdIns(3,4,5)P-3, a product of PtdIns 3-kinase. Stimulatio n of the GTP-binding-protein-linked receptor by N-formylmethionyl-leuc yl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5) P-3 in the cells. The effect of insulin was, while that of fMLP was no t, accompanied by increased PtdIns 3-kinase activity in the anti-phosp hotyrosine immunoprecipitate. The combination of insulin and fMLP indu ced more PtdIns(3,4,5)P-3 production than the sum of the individual ef fects. The insulin-induced recruitment of PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate was unaffected by the combi ned treatment with fMLP. To investigate the mechanism underlying the s ynergistic accumulation of PtdIns(3,4,5)P-3, we separated the cytosoli c proteins of THP-I cells on a Mono Q column. PtdIns 3-kinase activiti es were eluted in two peaks, and one of the peaks markedly increased o n the addition of beta gamma-subunits of GTP-binding proteins (G beta gamma). The other peak was affected only slightly by G beta gamma, but was synergistically increased by G beta gamma and a tyrosine-phosphor ylated peptide which was synthesized according to the amino acid seque nce of insulin receptor substrate-1. The activity in the latter fracti on was completely immunoprecipitated by an antibody against the regula tory subunit of PtdIns 3-kinase (p85). These results suggest that the conventional PtdIns 3-kinase (p85/p110), which has been implicated in insulin-induced cellular events, or a closely related isoenzyme is con trolled by a combination of a tyrosine-phosphorylated protein and a GT P-binding protein in intact cells.